US7790173B2 - Pharmaceutical compound capable of induce immune protective response against Dengue virus having the capsid protein of the Dengue virus - Google Patents

Pharmaceutical compound capable of induce immune protective response against Dengue virus having the capsid protein of the Dengue virus Download PDF

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US7790173B2
US7790173B2 US12/067,129 US6712906A US7790173B2 US 7790173 B2 US7790173 B2 US 7790173B2 US 6712906 A US6712906 A US 6712906A US 7790173 B2 US7790173 B2 US 7790173B2
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capsid protein
dengue virus
dengue
protein
virus type
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US20080311157A1 (en
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Laura Lazo Vázquez
Lisset Hermida Cruz
Carlos López Abarrategui
Beatriz de la Caridad Sierra Vázquez
Susana Vázquez Ramundo
Iris Valdez Prado
Gerardo Enrique Guillen Nieto
Maria Guadalupe Guzmán Tirado
Aida Zulueta Morales
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Centro de Ingenieria Genetica y Biotecnologia CIGB
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/295—Polyvalent viral antigens; Mixtures of viral and bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/18—Togaviridae; Flaviviridae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention is related to the field of biotechnology and the pharmaceutical industry, in particular to the obtaining of proteins capable of inducing an immune response against the infection with Dengue virus, quoted from now on as DEN, while avoiding the antibody-dependent enhancement phenomenon that has been described in persons re-infected with this virus.
  • the causal agent of this disease is the Dengue virus of the genus Flavivirus , family Flaviviridae, which is transmitted by the mosquito Aedes aegypti (Leyssen P., De Clerco E., Neyts J. 2000. Perspectives for the treatment of infections with Flaviviridae. Clin. Microbiol. Rev. 13: 67-82).
  • Dengue virus is an RNA positive coated virus, whose genome contains only one reading frame. This RNA is translated in a polyprotein that is processed in three structural proteins and seven non-structural proteins. (Russell P. K., Brandt W. E., Dalrymple J. M. 1980. Chemical and antigenic structure of flaviviruses. The togaviruses: biology, structure, replication. Schelesinger R. W. (ed.). 503-529).
  • the antibody-virus complexes can be internalized by cells presenting Fey receptors in the membranes, like monocytes and macrophages. This mechanism, known as antibodies-dependent enhancement (ADE) occurs during secondary infections.
  • AD antibodies-dependent enhancement
  • Halstead et al. (Halstead S. B., Scanlon J. E., Umpaivit P., Udomsakdi S. 1969. Dengue and Chikungunya virus infection in man in Thailand, 1962-1964. IV. Epidemiologic studies in the Bangkok metropolitan area. Am. J. Trop. Med. Hyg. 18: 997-1021.), in a 3-year study in Bangkok, Thailand, reported that the hospitalization indices by DEN infection among children, reached a maximum in those between 7 and 8 months old. These indices were four to eight times greater than the observed between children of 1-3 months and twice than that in children of 3 years. Kliks et al. (Kliks S.
  • T-cells epitopes have been reported mainly in nonstructural proteins (Kurane I, Zeng L, Brinton M A, Ennis F A. 1998. Definition of an epitope on NS3 recognized by human CD4+ cytotoxic T lymphocyte clones cross-reactive for dengue virus types 2, 3, and 4. Virology. 1998 Jan. 20; 240(2):169-74), but also are present in the envelope and in the capsid proteins (Bukowski, J. F., I. Kurane, C.-J. Lai, M. Bray, B. Falgout, and F. A. Ennis. 1989. Dengue virus-specific cross-reactive CD8 human cytotoxic T lymphocytes. J. Virol.
  • the capsid protein of Dengue virus has a molecular weight of 9 to 12 kDa (112-127 amino acids) and has a marked basic character because the 25% of its amino acids are arginine and lysine. The presence of these amino acids could favor antigenic presentations to the immune system due to the capacity of polycationic peptides to do so.
  • Poly-1-arginine synergizes with oligodeoxynucleotides containing CpG-motifs (CpG-ODN) for enhanced and prolonged immune responses and prevents the CpG-ODN-induced systemic release of pro-inflammatory cytokines.
  • Vaccine. 20: 3498-3508 The protein is located totally within the virion structure without any exposed region (Kuhn R J, Zhang W, Rossmann M G, Pletnev S V, Corver J, Lenches E, Jones C T, Mukhopadhyay S, Chipman P R, Strauss E G, Baker T S, Strauss J H. 2002. Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell. March 8: 108(5):717-25).
  • Flavivirus Capsid Is a Dimeric Alpha-Helical Protein. Journal of Virology, p 7143-7149, Vol. 77, No. 12) purified the capsid protein of VD2 obtained by the recombinant way in Escherichia coli ( E. coli ) and demonstrated that this protein behaves like a dimmer in solution without nucleic acids. Its secondary structure is mainly in form of alpha-helices and is composed by four of these helices, being one of those of greater length in the C-terminal end. The N-terminal end does not present a defined structure and its deletion does not affect the structural integrity of the protein.
  • This invention describes for the first time that the capsid of DEN-2 virus, obtained by a recombinant way in E. coli and with only a 40% of purity, is able to induce a protective immune response against the challenge with lethal DEN-2 virus in mice. It was demonstrated that this highly purified protein, retained its protective capacity, which was surpassed in the immunization of mice with the particulated form of the molecule. Moreover, it was demonstrated that the reached protection was mediated by CD8+ T-cells, a novel element considering that the reported T-cells epitopes for the capsid so far, are recognized by CD4+ T cells (Gagnon S J, Zeng W, Kurane I, Ennis F A. 1996.
  • this recombinant molecule was mixed with the PD5 protein, which is formed by the P64k protein of Neisseria meningitidis and the III domain of the envelope protein of the Dengue-2 virus.
  • This fusion protein is able to generate a highly serotype specific, protective and neutralizing immune response, with a low probability of generating the phenomenon of antibodies dependent enhancement (Hermida L, Rodriguez R, Lazo L, Silva R, Zulueta A, Chinea G, Lopez C, Guzman M G, Guillen G. 2004.
  • a dengue-2 Envelope fragment inserted within the structure of the P64k meningococcal protein carrier enables a functional immune response against the virus in mice. J Virol Methods. 2004 January; 115(1):41-9).
  • the objective of this invention is to obtain a recombinant protein corresponding to the capsid protein of Dengue virus, which generates a protective response against the infection with the lethal virus when is inoculated in mice.
  • the gene codifying for the capsid protein of Dengue virus was inserted into a plasmid containing the phage T5 promoter.
  • This protein was purified approximately till a 40% of purity, and was adjuvated in aluminum hydroxide to be inoculated in Balb/c mice. A month upon the last dose the antiviral antibody response was measured. At the same time the lymphoproliferative response in spleens stimulated in vitro with the dengue virus was determined. As a result no antiviral antibodies were induced while a significant lymphoproliferative response was detected. In parallel, in not bleeding mice, the protection assay was done. A lethal doses corresponding to 100 LD 50 of Dengue virus was inoculated, the disease symptoms and death were observed during 21 days. As a result a 44% of survival-immunized mice were obtained while in the negative control group all mice died. This is the first evidence of a protective response against Dengue virus by the immunization only with the capsid protein.
  • the semipurified preparation was detected a fraction with lower retention times, while in the purified sample a retention time corresponding to the dimeric form of the molecule was detected.
  • the dimeric and particulated preparations both with more than 95% of purity, were inoculated in mice.
  • the dimeric preparation was adjuvated with Freund Adjuvant and aluminum hydroxide, while the particulated variant was adjuvated only with aluminum hydroxide.
  • a dengue-2 Envelope fragment inserted within the structure of the P64k meningococcal protein carrier enables a functional immune response against the virus in mice. J Virol Methods. 2004 January; 115(1):41-9).
  • a lymphoproliferative response higher than that induced only by the capsid and significantly higher than that induced by the fusion protein was detected.
  • a plasmid containing the III domain of the envelope protein of DEN-2 virus fused to the N-terminal of the capsid protein gene was constructed.
  • the resulting protein with a 40% of purity, generated in Balb/c mice a lymphoproliferative response higher than that induced by the capsid alone and a serotype specific antibodies response higher than that induced by PD5.
  • FIG. 1 Cloning strategy of the capsid protein of DEN-2 virus to generate PDC-2.
  • DEN2 C Fragment of the capsid protein of DEN-2.
  • FIG. 2 Analysis by SDS-PAGE at 15% of the PDC-2 semipurification process. 1. Rupture supernatant. 2 and 3. Fraction not adsorbed to Q Sepharose FF. 4. Fraction eluted with NaCl 1M.
  • FIG. 3 Analysis by SDS-PAGE at 15% of the PDC-2 purification process. 1. Rupture supernatant, 2. Fraction not absorbed to the gel, 3. Washed (350 mM NaCl), 4. Eluted fraction (750 mM NaCl), 5. Fraction in Tris 10 mM, EDTA 1 mM.
  • FIG. 4 Chromatographic profile in Superdex 200 of the semipurified (A) and pure (B) preparations of PDC-2.
  • FIG. 5 Electronic microscopy pictures of the pure PDC-2 preparation before (A) and after (B) the treatment with oligonucleotides.
  • FIG. 6 Cloning strategy of the capsid protein of DEN-1 virus to generate PDC-1.
  • DEN1 C Fragment of the capsid protein of DEN-1.
  • FIG. 7 Analysis by SDS-PAGE at 15% of the PDC-1 semipurification process. 1. Molecular weight marker. 2. Rupture supernatant 3. Fraction not adsorbed to Q Sepharose FF.
  • nucleotide sequence that codes for amino acids 1 to 99 of the capsid protein from DEN-2 virus (Sequence No. 3), was amplified with the oligonucleotides identified in the sequence list as Sequence No. 1 and Sequence No. 2 from the DEN-2 virus strain genotype Jamaica (Deubel V., Kinney R. M., Trent D. W. Nucleotide sequence and deduced amino acid sequence of the nonstructural proteins of Dengue type 2 virus, Jamaica genotype: Comparative analysis of the full-length genome. Virology 1988.165:234-244).
  • the vector was created by digestion of the plasmid pQE-30 with BamHI/HindIII, which contains the phage 15 promoter and a 6-histidine tail in the N-terminal region (Sequence No. 6). Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions.
  • Competent cells XL-1 Blue (Hanahan D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557-580) were transformed with the selected clone called pDC-2 ( FIG. 1 and Sequence No. 4).
  • the transformed E. coli strains were cultivated in Luria Bertani medium (LB) supplemented with Ampicilline 50 ⁇ g/mL for 10 h at 37° C. Isopropyl-B-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM was used for the induction of the promoter. Upon growing the colony, an SDS-PAGE of the cellular lysate was done. As a result, a 15-kDA band was obtained.
  • the protein was recognized by an anti-DEN-2 hyperimmune ascitic fluid (HMAF). This protein was denominated PDC-2 (Sequence No. 5).
  • the biomass obtained from the E. coli strain transformed with pDC-2 and grown at 37° C. was disrupted by French press.
  • the recombinant protein was obtained equally distributed between the soluble and insoluble fractions.
  • the soluble fraction was subjected to an anionic interchange chromatography, using a Q Sepharose FF column and the buffer Tris 10 mM pH 8.
  • the protein in the non-absorbed fraction was obtained with 40% purity and was used for the immunological studies ( FIG. 2 ).
  • mice Three groups of 30 Balb/c mice were used. Two of them were immunized with 10 ug of the recombinant protein by intraperitoneal route, using Freund's Adjuvant (FA) in one of the groups and aluminum hydroxide in the other. The soluble fraction resulting from the rupture of the pQE-30-transformed cells was used as negative control adjuvanted with FA; 10 animals were bled 15 days after the third dose and the antibody titers against DEN-2 were determined by ELISA. After the immunization with the recombinant protein, formulated in either adjuvant, no antibody titers were obtained.
  • FA Freund's Adjuvant
  • mice were used from each of the groups immunized with the recombinant protein adsorbed in aluminum hydroxide and with the control preparation. Each animal received a dose of 100 LD 50 of lethal DEN-2 virus by intracranial inoculation and was observed for 21 days to obtain the percentages of lethality in terms of death by viral encephalitis.
  • a positive control a group of 10 mice immunized with infective DEN-2 virus (10 4 pfu) was used. All mice in the positive control group survived, while in the negative control group all mice were sick by day 7-11 after challenge and 100% mortality was obtained by day 21. Finally, the group immunized with the recombinant protein PDC-2 presented 44.4% protection (table 2).
  • the biomass obtained from the E. coli strain transformed with pDC-2 and grown at 37° C. was disrupted by French press.
  • the recombinant protein was obtained equally distributed between the soluble and insoluble fractions.
  • the soluble fraction was subjected to a cationic interchange chromatography, using an SP-Sepharose FF column and the buffer Tris 10 mM, Tween 0.5%, urea 7M, pH 8.
  • the column was washed with buffer diethanolamine 30 mM, NaCl 350 mM, pH 10.3.
  • the elution of the protein of interest was done with buffer diethanolamine 30 mM, NaCl 750 mM, pH 10.3.
  • the buffer was exchanged using G-25 columns.
  • the protein was obtained with 96% purity in buffer Tris 10 mM, EDTA 1 mM ( FIG. 3 ).
  • the buffer was exchanged to Hepes 25 mM, KAc 100 mM, MgAc2 1.7 mM, pH 7.4. After heating the protein and the mixture of oligonucleotides for 1 min at 37° C., they were incubated in an equal volume for 30 min at 30° C. As a negative control of the experiment, the protein was incubated without the oligonucleotides. When both preparations were observed with an electron microscope, a large quantity of particles of approximately 21 nm diameter, were observed in the sample of protein previously incubated with the mixture of oligonucleotides, while in the control sample no particles were observed ( FIG. 5 ).
  • mice Five groups of 20 Balb/c mice were used. Two of them were immunized with 10 ug of the dimeric purified recombinant protein by intraperitoneal route, using aluminum hydroxide and Freund's adjuvant. Another group was immunized with 10 ug of the purified and particulated capsid protein adjuvanted with aluminum hydroxide. The soluble fraction from the rupture of XL-1 blue cells transformed with the plasmid pQE-30 and subjected to the same purification steps than PDC-2 was used as negative control, adjuvanted with Freund's adjuvant. The fifth group was immunized with DEN-2 virus as positive control.
  • mice from each group received a dose of 100 LD 50 of lethal DEN-2 by intracranial inoculation and were observed for 21 days to obtain the percentages of survival. All mice in the positive control group survived, while in the negative control group all mice were sick by day 7-11 after challenge and 0% mortality was obtained. Finally, from the groups immunized with the recombinant protein, the group immunized with pure dimeric PDC-2 presented a 20% protection when immunized with aluminum hydroxide and a 40% protection when Freund's adjuvant was used. Additionally, in the group that received the reparticulated pure protein adjuvanted with aluminum hydroxide. 90% of mice were protected (Table 4).
  • Twenty animals were inoculated with the mixture of 10 ug of the particulated pure capsid protein and 20 ug of protein PD5 (Sequence No. 23) in three doses spaced fifteen days apart.
  • aluminum hydroxide was used as adjuvant.
  • the animals were bled and the sera tested for antiviral antibodies by ELISA.
  • the group immunized with the mixture developed serotype-specific antibodies with titers higher than those of the group immunized only with protein PD5 and, at the same time, titers in these two groups were higher than those in the group immunized with protein PDC-2, where no Abs against DEN-2 virus were detected.
  • 10 additional animals were taken from each group for lymphoproliferation assays. The cells from the spleens of these animals were extracted and stimulated with the infective DEN-2 virus.
  • the stimulation indexes were higher than those in the group immunized with the capsid protein only. The lowest stimulation indexes were obtained in the group immunized with protein PD5.
  • the reparticulated and the dimeric capsid proteins were inoculated in Balb/c mice to obtain some evidence of induction of cellular immune response.
  • the nucleotide sequence that codes for amino acids 1 to 100 of the capsid protein of DEN-1 virus was amplified with the oligonucleotides identified in the sequence list as Sequence No. 8 and Sequence No. 10 from the DEN-1 viral strain.
  • the vector was generated by digestion BamHI/HindIII of the plasmid pQE-30, which contains the phage T5 promoter and a 6 histidine tail in the N-terminal region (Sequence No. 6). Upon ligation, the recombinants were analyzed by restriction and the positives clones were sequenced to check the junctions. Competent cells XL-1 Blue (Hanahan D. 1983.
  • the biomass obtained from the E. coli strain transformed with pDC-1 and grown at 37° C. was disrupted by French press.
  • the recombinant protein was obtained equally distributed between the soluble and insoluble fractions.
  • From the soluble fraction an anionic interchange chromatography was done, using a Q Sepharose FF column and the buffer Tris 10 mM pH 8.
  • the protein in the non-absorbed fraction was obtained with 45% of purity, and was used to the immunological studies.
  • mice Two groups of 30 Balb/c mice were used. One of them was immunized with 10 ug of the recombinant protein by intraperitoneal route, using the aluminum hydroxide as adjuvant. The soluble fraction resulting from the rupture of the pQE-30-transformed cells adjuvanted with aluminum hydroxide was used as negative control. A part of the animals (10 mice) were bled 15 days after the third dose and the antibody titers against DEN-1 were determined by ELISA. After the immunization with the recombinant protein, no antiviral antibody titers were obtained.
  • Antibodies titers against DEN-1 virus from sera obtained after the immunization with the semipurified PDC-1 Anti- DEN-1 ELISA titers XL-1 blue Mouse Control ( ⁇ ) PDC-1 1 ⁇ 1:100 ⁇ 1:100 2 ⁇ 1:100 ⁇ 1:100 3 ⁇ 1:100 ⁇ 1:100 4 ⁇ 1:100 ⁇ 1:100 5 ⁇ 1:100 ⁇ 1:100 6 ⁇ 1:100 ⁇ 1:100 7 ⁇ 1:100 ⁇ 1:100 8 ⁇ 1:100 ⁇ 1:100 9 ⁇ 1:100 ⁇ 1:100 10 ⁇ 1:100 ⁇ 1:100 ⁇ 1:100
  • mice were used from each of the groups immunized with the recombinant protein adsorbed in aluminum hydroxide and with the control preparation. Each animal received a dose of 100 LD 50 of lethal DEN-1 by intracranial inoculation and was observed for 21 days to obtain the percentages of lethality in terms of death by viral encephalitis.
  • a positive control a group of 10 mice immunized with infective DEN-1 virus (10 4 pfu) was used. All mice in the positive control group survived, while in the negative control group all mice were sick at day 7-11 after challenge and 100% mortality was obtained at day 21. Finally, the group immunized with the recombinant protein PDC-1 presented 50% of protection (Table 11).
  • nucleotide sequence that codes for amino acids 286 to 426 of the envelope protein from DEN-2 (Sequence No. 12), corresponding to the region of the domain 111 of the protein, was amplified with the oligonucleotides identified in the sequence list as Sequence No. 13 and Sequence No. 14 from the DEN-2 virus strain genotype Jamaica (Deubel V., Kinney R. M., Trent D. W. Nucleotide sequence and deduced amino acid sequence of the nonstructural proteins of Dengue type 2 virus, Jamaica genotype: Comparative analysis of the full-length genome. Virology 1988.165:234-244).
  • the vector was created by digestion of the plasmid pDC-2 with BamHI/BamHI, which contains the phage T5 promoter, a 6-histidine tail in the N-terminal region and the region corresponding to 100 amino acids of the capsid protein of DEN-2 virus. Upon ligation, the potential recombinants were analyzed by restriction enzyme digestion and positive clones were sequenced to check up the junctions. Finally the clone selected was named pDC-2 Dom III (Sequence No 15).
  • Competent cells XL-1 Blue (Hanahan D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557-580) were transformed with the selected clone called pDC-2 DomIII.
  • the E. coli strains transformed were cultivated in LB supplemented with Ampicilline 50 ⁇ g/mL for 10 h at 37° C. isopropyl-B-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM was used to the induction of the promoter. Upon growing the colony, an SDS-PAGE of the cellular lysate was done. As a result, a 30-kDA band was obtained.
  • the protein was recognized by an anti-DEN-2 HMAF. This protein was denominated PDC-2 Dom III (Sequence No. 16).
  • the biomass obtained from the E. coli strain transformed with pDC-2 DomIII and grown at 37° C. was disrupted by French press.
  • the recombinant protein was obtained equally distributed between the soluble and insoluble fractions.
  • From the soluble fraction an anionic interchange chromatography was done, using a Q Sepharose FF column and the buffer Tris 10 mM pH 8.
  • the protein in the non-absorbed fraction was obtained with 40% of purity, and was used to the immunological studies ( FIG. 2 ).
  • mice Five groups of 30 Balb/c mice were used. One of the groups was immunized with 10 ug of the recombinant protein by intraperitoneal route, using aluminum hydroxide as adjuvant. The soluble fraction resulting from the rupture of the XL-1 Blue cells transformed with the plasmid pQE-30 was used as negative control, adjuvanted with aluminum hydroxide. Another two groups were included as controls. One of them was immunized with the protein PDC-2 and the other with the protein PD5 (this protein contains the domain III region of the envelope protein of DEN-2 virus). Ten animals from each group were bled 15 days after the third dose and the antibody titers against DEN-2 were determined by ELISA.
  • Sequence Listing is disclosed on a computer-readable ASCII text file titled, “sequence_listing.txt”, created on Jul. 23, 2008.
  • sequence_listing.txt file is 44 kb in size.

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Cited By (4)

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US20110081646A1 (en) * 2009-08-31 2011-04-07 Gen-Probe Incorporated Dengue virus assay
US8999675B2 (en) 2009-08-31 2015-04-07 Gen-Probe Incorporated Dengue virus assay
US20150328304A1 (en) * 2012-12-27 2015-11-19 Centro De Ingeniería Genética Y Biotecnología Dengue virus vaccine composition
US9463235B2 (en) * 2012-12-27 2016-10-11 Centro De Ingeniería Geneética Y Biotechnología Dengue virus vaccine composition

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